By coupling the unique model system of the mammalian X chromosome with powerful new ways to probe the organization of specific genes. RNAs, or chromosomes, the work proposed here has potential to advance our understanding of higher-level chromosome and nuclear structure, in general, and its relationship to transcription and X inactivation, in particular. We will systematically investigate and compare specific aspects of the molecular cytology of Xi (inactive X) and Xa (active X), in the normal native state, in naturally occurring chromosomal rearrangements. and in genetically manipulated cells. Experiments proposed largely relate to areas of recent developments in nuclear structure, each of which has fundamental significance and is supported by strong preliminary studies. The first concerns emerging evidence for nuclear compartmentalization. We hypothesize that the genome, as embodied by interphase chromosomes, is non randomly integrated in a gene specific manner with respect to splicing factor/poly A RNA rich "compartments" or "domains", specialized for a function(s) related to RNA metabolism. We further suggest that this spatial arrangement related to gene expression. Specific aim 1 compares euchromatic Xa versus heterochromatic Xi, including genes which escape inactivation, to address aspects of this and related ideas. Analysis of the important Fragile X region is included where chromosome structure is known to be perturbed by a trinucleotide repeat expansion. The second recent breakthrough concerns XIST a gene strongly implicated in X inactivation. We hypothesize that RNA from the XIST gene is itself involved in inactivation. Preliminary studies suggest that this RNA may provide a precedent for RNA involvement chromosome and nuclear structure. The molecular cytology of this RNA becomes key to understanding the structure/function relationship. Aim 2 proposes to examine the RNA distribution relative to Xi DNA sequences on normal and rearranged chromosomes, by both 2-D and detailed 3-D analysis. Analysis of specific genes on Xi will contribute to the formation of a model for the interphase organization of genes which escape inactivation. Aim 3 will investigate the relationship of XlST RNA to Xi and nuclear structure in cells in which the RNA, or portions of it, are expressed from foreign chromosomal or nuclear context. Reciprocal transgenes, consisting of an autosomal gene translocated to Xi, will examine inter-relationships between XIST RNA. the splicing factor rich compartment, and underlying nuclear structure. Aim 4 will extend aspects of the above studies to immunofluorescence differences in protein content on Xi, Xa, an Xi:autosome translocations, for example to explore proteins known to bind XIST RNA, or to explore an innovative means to isolate pure populations of Xi and Xa as a resource for studies of chromatin.